Most mammalian tissues contain two prominent beta-glucosidases. One is lysosomal glucocerebrosidase (GC), the enzyme whose deficiency is responsible for Gaucher's disease, and that normally catalyzes the hydrolysis of glucocerebroside. The other is broad-specificity or cytosolic beta-glucosidase. It is as designated because of its subcellular location in liver and spleen and because it hydrolyzes a variety of glycosides, including beta-D-glucosides, beta-D-galactosides and alpha-L- arabinosides. The function of this second beta-glucosidase is obscure. However, its ability to degrade a variety of plant glycosides, including cyanogenic glucosides, suggests the enzyme may play a role in detoxication of xenobiotic compounds. The activity of GC is strongly dependent upon acidic lipids, a requirement that is best satisfied by phosphatidylserine (PS) or sulfatide (S). Furthermore, assay of GC activity in the presence of phosphatidylserine or sulfatide allows one readily to differentiate GC of patients with nonneuropathic (type 1) and neuropathic (type 2) Gaucher's disease. Control and type 2 GCs respond to an 11,000 dalton heat-stable factor (HSF) isolated from Gaucher spleen whereas the type 1 enzyme does not. The overall goal is to investigate the structure and function of the two beta-glucosidases. First, the interaction between human spleen and brain GC and activator lipids incorporated into unilamellar vesicles and Triton X-100 micelles of known composition will be investigated by physical- chemical and kinetic approaches. Specifically, we will address the issues of lipid-enzyme stoichiometry and specificity, size of the active enzyme, and the mechanism of activation. The structure and function of the HSF will be further characterized by sequence analysis and high resolution 1H- NMR spectroscopy. For the broad-specificity beta-glucosidase, kinetic studies will be performed to further define the range of plant glycosides that either serve as substrates or which inhibit the enzyme. Experiments are also planned which will characterize the interaction of the lipid binding site of the enzyme with physiologically relevant lipids (e.g., gangliosides). Finally, in an effort to compare the primary structures of the two beta- glucosidases, the sequence of the broad-specificity beta-glucosidase messenger RNA will be determined.